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DiscoveRx GPCR cell based assays for identifying novel ligands, studying receptor pharmacology and uncovering ligand bias.
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Revealing Ligand & GPCR Bias Using A Combination
of 2nd Messenger, β-Arrestin & Internalization
EFC Assay Formats
DiscoveRx – Your Drug Discovery Partner
Catalog Products
Profiling and Screening
Custom Assay
Development
Cell-Based Assays246 GPCR cell lines
15 Cell Based Kinases26 NHR cell lines6 Pathway assays
DiscoveRx Core Technology
Cell Biology
• Cellular GPCR Assays• Cellular NHR Assays• Cellular Kinase Assays • Cellular Protease Assays• Pathway / Signaling Assays
PathHunterTM Product Line
• GPCR Signaling Assays• Generic Kinase Assays• Protease Technology• Unactive Kinase Technology
HitHunterTM Product Line
Biochemical
+
Inactive Fragments Active Enzyme
PK PK
Active EnzymeEnzyme
Complementation
Evolution of Understanding GPCR Function
1.Binding - On/ Off Radioligand binding studies
2. Receptor Activation Through Ga Second messenger assays
3. Receptor De-sensitization via b-ArrestinInternalization & recruitment studies
Evolution of Understanding GPCR Function4. b-Arrestin involved in desensitization but also has signaling properties
Kinases (MAPK, P3K, AKT)
5. Novel conformations of the same receptor can initiate different signaling cascades Now understood that agonists can activate only a subset of signals
Balanced Signal
Biased Signals
Originally thought that all downstream pathways equally activated for a given agonist
Evolution of GPCRs
Functional GPCR Activities & Ligand Bias
Arrestin
Second MessengerSignaling
cAMP
Calcium
Internalization
Events following ligand binding to GPCRs
Differential activation of these pathways occurs using the
same ligand (Ligand Bias)
GPCR Activation: Multiple Biological Processes
cAMPCalciu
m
G-protein Activation
Arrestin Recruitment
Internalization
GRK
• 2nd Messenger Signaling
• Alternate pathway• Short term
desensitization• Contributes to
Internalization
• Long term desensitization
• Altered Trafficking• Degradation /
Recycling
Opportunity for ligand
bias
1
Long Acting Agonists
2
Avoid Unwanted Signaling
3
Functional Antagonists, Receptor removal
Monitoring each function can aid in compound characterization
PathHunter™ Protein Interaction Technology
+
Inactive Fragments Active Enzyme
PKPK
Active Enzyme
Weak/No activity High activity
EnzymeComplementation
+
Modified EnzymeComplementation
for Protein interactions
Features:• Generic, homogeneous approach applicable to a wide range of drug target classes• Flexible, small peptide tag with options for high and low affinity• Only technology successfully transitioned from biochemical to cell based format
GPCR Activation & b-Arrestin Binding
Small protein tag (42 aa) Dynamic protein interaction assay Target-specific signal Chemiluminescent or fluorescent
detection Transfers easily from benchtop to
full HTS campaigns10-11 10-10 10-9 10-8 10-7 10-6 10-50
10000
20000
30000
40000
50000
Isoproterenol [M]
RL
U
PathHunter™ b-Arrestin Assays
ADRB2
Arrestin-Based GPCR Signaling
10-1410-1310 -1210-1110-10 10-9 10-8 10-7 10-6 10-50
10000
20000
30000
40000
50000
Isoproterenol [M]
RL
U
SSTR2 (Gi) CHRM5 (Gq)
ADRB2 (Gs)
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -60
500
1000
1500
2000
2500
3000
3500
Somatostatin 28
RL
U
EC50=2.0 nM S/B=42 ACHR5
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-30
2000
4000
6000
8000
10000
12000
14000
Oxotremorine M
RL
U
EC50=3.1 μM; S/B=5.9
EC50=11.2 nM S/B=6.6
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
10000
20000
30000
40000
Glucagon [M]R
LU
EC50=15.3nM; S/B=11
Type B Glucagon (GCGR)
1 hr1 hr
Read Luminescence
1 hour
1 hour
Thaw & Plate Cells
12-48 hrs
Highlights of PathHunter™ Arrestin Format Stochiometric Signal
GenerationCompound EfficacyAntagonist Mode
Predictable Pharmacology
Specificity Serum samples
• Chemokines • Bioactive lipids • Antibody Supernatants
CHO A2 ADRB2 Clone Ligand Rank Order
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-30
250
500
750
1000
1250
1500
1750SalbutamolIsoproterenol(-) EpinephrineDeoxyepinephrineClenbuterolSalmeterolDobutamineFormoterolIsoetharineMetaproterenolRitodrineTerbutalineAgonist [M]
RL
U
CHO A2 GLP2R
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-50
2500
5000
7500
10000
GLP 2 (1-33) human [M]
RL
U
10-8 10-7 10-6 10-5 10-4 10-30
1000
2000
3000
4000
5000
6000
7000
8000
20% FBS40% FBS60% FBS80% FBS
Anti-CCL20 mAb [g/mL] + 10nM CCL20
RL
U
PathHunter Performance
Real-time analysis
Signal to noise
FormoterolClenbuterol
Isoproterenol
ADRB2
PathHunter clones (Receptors tested with
reference agonist)
S:B
Rati
o
>50% are 9X or better
Arrestin is G-protein Independent
G-protein inhibitor independent
CHO Arrestin FPRL1
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-50
10000
20000
30000
40000
50000
60000(+) Pertussis tox(-) Pertussis tox
WKYMVm-NH2 [M]
RL
U
CHO Gi FPRL1
10-14 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60
250
500
750
1000
1250
(+) Pertussis tox(-) Pertussis tox
WKYMVm-NH2 [M] + 20 M Forskolin
RL
U
cAMP Assay Arrestin Assay
Arrestin binding and internalization can occur in the absence of G-protein activity
CXCR7
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
10000
20000
30000
Complement C5a [M]
RL
Us
10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60
10000
20000
30000
40000
SDF1a [M]
RL
U
C5L2
Lambertus et al. J. Biomol Screening (2009)
Arrestin in the absence of G-protein activation
Basis for Arrestin Ligand Bias
Second Messenger
Native Gi, Gs, and Gq coupled cell lines HitHunter® biochemical reagents for cAMP detection Complex pharmacology- Agonists, antagonists, partial agonists & allosterism Differentiating compounds - Large assay windows, ideal for difficult Gi targets Miniaturizable Fluorescent and Aequorin-based calcium detection
10-13 10-12 10-11 10-10 10-9 10-8 10-70
1000
2000
3000
4000
Angiotensin II [M]
RL
U
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
1000
2000
3000
4000
[Sar1, Ile4,8]-Angiotensin II [M]
RL
U
10-15 10-14 10-13 10-12 10-11 10-10 10-9 10-80
250
500
750
1000
1250
1500
Angiotensin II [M]
RL
U
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
250
500
750
1000
1250
1500
[Sar1, Ile4,8]-Angiotensin II [M]
RL
U
Arrestin Biased Agonism
Biased agonist SII
Reference agonist
Biased agonist SII
Reference agonist
Ca++
AGTR1
10-1310-1210 -1110-10 10-9 10-8 10-7 10-6 10-5 10-40
500
1000
1500
2000
Ligand [M]
RL
U
GlucagonDHG
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
250
500
750
1000
1250
Ligand [M]
RL
U
Glucagon
DHG+ 300nM Glucagon
cAMP
GCGR
DHG is a ligand for the GCGR receptor-- antagonist in Arrestin but a partial agonist in
cAMP
PathHunter is Ideal for Investigating Ligand Bias
Lessons Learned
7TMR SignalingBoth G-protein dependent & independent
pathwaysLinked but not dependent pathwaysCan be modulated independently by
compounds
PathHunter™ Arrestin TechnologyBroadly applicable measure of arrestin
signalingIdeal for difficult targets and orphansHTS friendlyOpportunity for multiplexed measurements
GPCR Internalization
Applicable to the majority of GPCRs
Reduces the number of cell surface receptors acutelyRecycledTargeted for degradationFunctional antagonism
Compartmentalized signaling has been observed
EAEA
EA
Complemented Enzyme
Arr
esti
n R
ecru
itm
en
t
- Gain-of-signal, large S:B- Arrestin dependent-Short-term desensitization at membrane
PathHunter™ GPCR Internalization Assays:2 Convenient Platforms. Same Answer. Does the receptor internalize?
Endo
EAEA
EA
Complemented EnzymeA
cti
vate
d I
nte
rnaliza
tion
- Gain-of-signal, large S:B- Arrestin dependent- Long-term desensitization at endosome
EA
EA
10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60
250
500
750
1000
CCL3 [M]
RL
U
10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-30
250
500
750
Oxotremorine-M [M]
RL
U
CCR5 M5NTSR1
Neurotensin Chemokine Muscarinic
PathHunter™ Activated GPCR Internalization Assays:Broadly Applicable
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-50
2500
5000
7500
S/B = 47.0
BOTTOM TOP LOGEC50 HILLSLOPE EC50
NTSR1177.46785-8.8501.6511.4128e-009
Neurotensin [M]
RL
U
Tested across 35 different families Over 65 targets available (over 80 by
3/1) Building out specific panels
Compound selectivity Timing, magnitude, cell type
specificity
GPCR “Trio Project” For Ligand Bias
24 targets tested in all 3 formats, average of 5 agonists per target
Pharmacology differences observed in 11 of 24 Expected overlap between cAMP/Calcium and ArrestinOnly 1% of ligands are completely biased, 5-10%
show some biasMore differences with internalization
Currently expanding studies and following up on “biased” ligands
22
cAMP
0.00
010.
001
0.01 0.
1 1 10 100
1000
0
25
50
75
100
125CXCL1CXCL2CXCL3CXCL5CXCL6Interleukin-8
Agonist + 15 M Forskolin
% c
AM
P I
nh
ibit
ion
Internalization
0.00
010.
001
0.01 0.
1 1 10 100
1000
0
25
50
75
100
125
150CXCL1CXCL2CXCL3CXCL5CXCL6Interleukin-8
Agonist
% A
ctiv
atio
n
Arrestin
-4.0
0-3
.00
-2.0
0-1
.00
0.00
1.00
2.00
3.00
0
25
50
75
100
125
150CXCL1CXCL2CXCL3CXCL5CXCL6Interleukin-8
Agonist
% A
ctiv
atio
n
Arr
estin
No Bias detectedCXCR2
Inte
rnal
izat
ion
2nd M
esse
nger
DiscoveRx Trio Initiative
Arrestin
0.000
10.0
01 0.01 0.1 1 10 10
010
00
1000
0
0
25
50
75
100
125
150CCL3
CCL23
Agonist
% A
ctiv
atio
n
cAMP
0.000
10.0
01 0.01 0.1 1 10 10
010
00
1000
0
0
25
50
75
100
125
150CCL3CCL23
Agonist + 15 M Forskolin
% c
AM
P i
nh
ibit
ion
Internalization
0.000
10.0
01 0.01 0.1 1 10 10
010
00
1000
0
0
100
200
300
400
500
600CCL3
CCL23
Agonist
% A
ctiv
atio
n
CCR1Efficacy Bias
Arrestin
0.00
001
0.00
010.
001
0.01 0.
1 1 10 100
1000
1000
0
1000
00
0
25
50
75
100
125
150
Lys-BradykininBradykinin[Phe8"Psi"-BDK
Agonist
% A
ctiv
atio
n
Internalization
0.00
001
0.00
010.
001
0.01 0.
1 1 10 100
1000
1000
0
1000
00
0
25
50
75
100
125
Lys-BradykininBradykinin
[Phe8"Psi"-BDK
Agonist
% A
ctiv
atio
n
Calcium
0.00
001
0.00
010.
001
0.01 0.
1 1 10 100
1000
1000
0
1000
00
0
25
50
75
100
125
Lys-BradykininBradykinin[Phe8"Psi"BDK
Agonist
% A
ctiv
atio
n
Potency BiasBDK2
Uncover Biased Ligands Using the Complete DiscoveRx GPCR Portfolio
• Opioid system controls responses to pain - OPRD1 (hDOR) activation alleviates persistent pain - Desensitization leads to persistent pain• Receptor responds to endogenous & synthetic ligands• hDOR undergoes rapid internalization• Compound-specific differences in re-sensitization have been identified
Delta Opioid Receptor Case Study
Prolonged G-protein activation
ProlongedDesensitizationFunctional Antagonism
Biased Signaling
2nd Messenger Arrestin Internalization
SNC-80 Results in OPRD1 Desensitization
• SNC-80 showed 95% inhibition sustained for 30 minutes
• Faster & stronger desensitization vs endogenous enkephalins
• Fresh ligand could not restore signal
slower
•SNC-80 identified as a strongly internalizing compound
•Agonist recycling is compound-specific
SNC-80 Results in Prolonged OPRD1 Internalization
In vivo Effects of a Non-Internalizing Ligand
First Challenge
2nd Challeng
eARMS390 and SNC80 are full agonists by GTPgS
SNC80 –EC50: 122nMARMS390-EC50: 170nMSummary• SNC80 is a
strongly internalizing ligand
• SNC80 behaves as a functional antagonist in vivo
10-1410-1310 -1210-1110-10 10-9 10-8 10-7 10-6 10-50
5000
10000
15000
20000
25000
Agonist [M] + 20 M Forskolin
RL
U
2nd Messenger
10-1210-1110-10 10-9 10-8 10-7 10-6 10-5 10-4 10-30
50000
100000
150000
200000
Agonist [M]
RL
U
Arrestin
10-1210-1110 -10 10-9 10-8 10-7 10-6 10-5 10-4 10-30
500
1000
1500
2000
Agonist [M]
RL
U
Internalization
DAMGO is a weak agonist compared to SNC-80
DAMGO weakly recruits Arrestin
SNC-80 strong Arrestin response
SNC-80 strong Arrestin-mediated internalization vs DAMGO
Differential GPCR Biology: OPRD1
More Information - CCKAR
• Full agonist in calcium
• Similar in vivo half lives
• Why is a compound that is less potent, more active in vivo?
Relevant, Predictive, Informative
Arrestin
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
100000
200000
300000
400000sCCK8A-71623
[M]
RL
U
Endo
10-12 10-11 10-10 10-9 10 -8 10-7 10 -6 10-50
1000
2000
3000
4000
5000
6000
7000sCCK8A-71623
[M]
RLU
Working hypothesis: A-71623 has prolonged duration of action due to poor internalization.
2nd Messenger Arrestin Internalization
Potency Efficacy Potency Efficacy Potency Efficacy
Compound X 10nM Full 5nM Full 5nM Partial
Compound Y 50nM Full 35nM Partial 35nM Full
Compound Z 20nM Partial 100nM Full 100nM Full
Moving the industry…….
High Content Compound Analysis
Arrestin CHRM2
10-10.0 10-7.5 10-5.0 10-2.5 100.00
50000
100000
150000
[M]
RL
U
Internalization CHRM2
10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-20
2500
5000
7500
10000
12500
[M]
RL
U
cAMP CHRM2
10-10 10 -9 10 -8 10 -7 10-6 10-5 10-40
2500
5000
7500
10000
12500
[M]
RL
U
CHRM2
Uncover Biased Ligands Using the DiscoveRx Complete GPCR Offering: CHRM2
Oxo-M gives a functional response
Behaves as an agonist
Oxo-M shows weak b-Arrestin
recruitment
Oxo-M shows strong Arrestin-mediated internalization
Kinetensin behaves as a weak agonist
Kinetensin is a weak agonist, similar EC50s
Kinetensin results in weak internalization
Calcium NTSR1
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
50
100
150
200
250
300
350
[M]
RL
U
Arrestin NTSR1
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
250000
500000
750000
1000000
1250000
[M]
RL
U
Internalization NTSR1
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40
10000
20000
30000
40000
50000
60000
70000
[M]
RL
U
NTSR1
*Currently building out data sets and algorithms (in collaboration) for Ligand Bias quantification*
Uncover Biased Ligands Using the DiscoveRx Complete GPCR Offering: NTSR1
Why Are PathHunter Internalization Assays Such An Important Drug Discovery Tool?• Receptor Internalization Influences Agonist Efficacy- Fewer receptors at the cell surface, reduces potency- Selective and non-selective agonists can be compared
• New classes of drugs can be developed - Functional Antagonists that remove receptors from cell
surface can reduce unwanted side effects-Non-recycling receptors may be better targets for chronic diseases (multiple administrations)
• Molecular Pharmacology - Determine a direct link between
internalization/localization & function for a target
DiscoveRx GPCR Portfolio Summary
Multi-Mode GPCR characterization provides: Extensive ligand analysis – identification of biased
ligands Potential to select ligands with specific properties and
durations Correlate biological function with biochemical
characterization Similar Studies
• OPRM1 - μ- Opioid receptors: correlation of agonist efficacy for signalling with ability to activate internalization: McPherson et al. Mol Pharm July 2010
• EP4 - Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands: Leduc et al. JPET July 2009
Prolonged G-protein activation
ProlongedDesensitizationFunctional Antagonism
Biased Signaling
2nd Messenger Arrestin Internalization
Biased Ligand Discovery with DiscoveRx
Monitor GPCR activity through multiple 7TMR signaling pathways and uncover novel, biased ligands
• Largest GPCR menu covering all 7TMR signaling pathways -- 2nd messenger, arrestin & internalization)
• Preferred supplier offering all technology platforms in the same robust, easy-to-use, HTS-friendly format
• Provide “Next Generation” GPCR platforms that can be used explore novel receptor biology including internalization and dimerization
DiscoveRx Comprehensive GPCR Portfolio
GPCR Pathway
DRX Products Advantages
Gi/Gs Native Gi/Gs cell lines,HitHunter™ cAMP XS+
Native, non-force coupled cell lines with chemiluminescent detection
Gq Native Gq cell lines,HitHunter™ Calcium No Wash PLUS
Native, non-force coupled cell lines with fluorescent detection
Gq HitHunter™ IP3 Secondary confirmation of Calcium response
G-protein Independent Signaling
PathHunter™ Arrestin De-orphanization, uncover novel pharmacology biased ligand discovery
PathHunter™ Activated GPCR Internalization Assays
Monitor activated GPCR internalization without imaging, biased ligand discovery
PathHunter™ GPCR Dimerization Assays
Specific and quantitative measure of GPCR heterodimers
Thank You!